(D-132) F-Actin Bundles and Cross-linkers in Osteocyte Dendrites

Introduction:: Osteocytes are the master orchestrators of bone remodeling, and their extensive dendrite network enables them to sense the external mechanical signals and communication with other cells.1,2 With aging, the dendrites deteriorate with bone quality declined.3 Similar to epithelial microvilli and inner ear stereocilia4, osteocyte dendrites are thought to contain rigid tightly packed cross-linked F-actin bundles as in previous mechanosensing models.5 However, the dendrite cytoskeleton details have not been revealed experimentally due to their entombment in a mineralized bone matrix and the high resolution needed to resolve the F-actin fibers (5-7 nm diameter). The goal of this study is to optimize protocols for high-resolution STED imaging and transmission electron microscopy (TEM) of the dendrite cytoskeleton of cultured osteocytes.
sub-track: Cellular and Molecular Biomechanics: Mechanobiology

Materials and Methods::

MLO-Y4 cells were seeded at a density of 4,000 cells/cm2 onto collagen-coated coverglass and cultured in alpha-MEM with 2.5% fetal bovine serum and 2.5% calf serum for 2-4 days. The F-actin cytoskeleton and selected cross-linkers were stained with SiR-Actin (in live cells), phalloidin and fimbrin and actinin antibodies (in 4% paraformaldehyde fixed cells), followed with STED (pixel ~16.5 nm) and confocal imaging (pixel ~100-200 nm). Additional TEM images (pixel ~0.55 nm) were also taken of cells on coverslips, fixed with 1% osmium tetroxide and 1% glutaraldehyde with an overnight 0.5% uranyl acetate staining and embedding in resin.

Results, Conclusions, and Discussions:: In cultured osteocytes, there were a few long cellular protrusions containing linear or curved F-actin fibers in a semi-parallel fashion. These fibers were neither located at the center (under confocal) nor formed compact bundles (observed under STED and TEM). Under STED (Fig. 1), fimbrin and actinin were found along the F-actin fibers and they were not fully co-localized with F-actin fibers in the dendrites as suggested in a previous study.6 Overall, the F-actin cytoskeleton of 2D cultured osteocytes is arranged differently than the previous linear actin core model. To understand osteocyte dendrites in vivo, better models are needed.

Acknowledgements (Optional): : Partially funded by NIH grants: P20 GM139760, P20 GM103446

References (Optional): :

(1) Schaffler, Calcif Tissue Int, 2014 (2) Bonewald, JBMR, 2011 (3) Tiede-Lewis, AGING, 2017 (4) Pelaseyed, J Cell Sci, 2018 (5) Wang, PNAS, 2007 (6) Murshid, J Bone Miner Metab, 2007